1. Volume 19, Issue 2, Pages 287-296 (February 2012)
ATP-Independent Control of Autotransporter Virulence Protein Transport via the Folding Properties of the Secreted Protein 
Jonathan P. Renn, Mirco Junker, Richard N. Besingi, Esther Braselmann, Patricia L. Clark 
Chemistry & Biology 
Volume 19, Issue 2, Pages (February 2012)
DOI: /j.chembiol
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2. Chemistry & Biology 2012 19, 287-296DOI: (10. 1016/j. chembiol. 2011
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3. Figure 1 Structural Organization of AT Passenger Domains and DHFR
(A–C) Crystal structure of hemoglobin protease passenger (PDB ID: 1WXR), a homolog of Pet (26% identical, 43% similar) (A), E. coli dihydrofolate reductase (PDB ID: 7DFR) (B), and pertactin passenger (PDB ID: 1DAB) (C). The stable core structures of Pet and pertactin are shown in blue, with other β-helical structure in green. Regions associated with function (Pet protease domain and proposed pertactin integrin binding loop) are shown in red, and other non-β-helical structure is in yellow.
(D) Schematic representation of the sizes of passenger domain structures and C-terminal stable cores for constructs expressed in this study. The N-terminal signal sequence and C-terminal 30 kDa β-domain porin of each preprotein construct are indicated by dashed lines.
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4. Figure 2
DHFR Stabilization by Methotrexate Blocks OM Secretion of DHFR-Petβ-Helix
(A) Methotrexate (MTX) does not inhibit secretion of wild-type Pet into the spent media (>100 kDa band, left), but does reduce secretion of DHFR-Petβ-helix chimera (<100 kDa band, middle). A band corresponding to the unprocessed DHFR-Petβ-helix precursor is detectable in whole cell lysates (130 kDa, closed circle, right); moreover, in the presence of MTX, a band corresponding to the size of the DHFR chimera periplasmic precursor was detected in whole cell lysates (open circle, right). Western blot visualized using an anti-Pet polyclonal antibody; note that removal of the Pet protease domain deletes some pAb binding epitopes in the DHFR-Petβ-helix passenger (Renn and Clark, 2008), which prevents direct comparison of band intensity to that of wild-type Pet passenger.
(B) OM secretion of the DHFR-Petβ-helix chimera is inhibited by MTX in a dose-dependent manner. Black circles: chimera bearing wild-type DHFR. Red diamonds: chimera bearing DHFR-I155A, which reduces the stability of DHFR (Table S1), requires higher concentrations of MTX to block secretion. Error bars represent the SD of three independent experiments.
(C) E. coli cultures were preincubated with fluorescein-labeled MTX before expression of uncleavable mutants of DHFR-Petβ-helix (N1018G/N1019I; Navarro-García et al., 2001), or the empty vector. MTX-FL stabilizes DHFR in the periplasm, blocking OM secretion of the passenger N terminus and rendering MTX-FL inaccessible to an OM impermeable quencher. Conversely, DHFR mutant I155A is destabilized, which increases its OM secretion efficiency (Figure 4), and enables quenching of MTX-FL bound to DHFR at the cell surface. Left panel, bright field images of E. coli bearing the indicated expression plasmid. Right panel, fluorescein fluorescence images of the same field. Scale bar represents 5 μm. See also Figure S1.
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5. Figure 3
OM Secretion of DHFR-Petβ-Helix Is Dependent on the Stability of DFHR
The amount of secreted protein was detected in the spent culture media using an anti-Pet passenger polyclonal antibody, normalized to wild-type DHFR (green), and plotted versus the free energy of unfolding of the DHFR domain (measured previously) (Perry et al., 1987; Garvey and Matthews, 1989; Arai et al., 2003; see also Table S1). The double mutation with ΔGfolding > 0 kJ/mol (Figure S1A) is shown in red; the red arrow indicates ΔGfolding > 0 kJ/mol. The linear fit (line) resulted in R = For comparison, secretion of PetΔprotease, which lacks an N-terminal globular domain, is represented as a horizontal blue line. Error bars represent the SD of at least four replicates. See also Figure S2.
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6. Figure 4
N-Terminally Stabilized DHFR-Petβ-Helix Accumulates at the E. coli Cell Surface
Immunofluorescence microscopy images of E. coli expressing empty vector (A), an uncleavable PetΔprotease mutant (N1018G/N1019I) that acts as a positive control for surface display of the Pet passenger (Navarro-García et al., 2001) and preserves Pet epitopes recognized by the anti-Pet passenger domain polyclonal antibody in the DHFR chimera constructs (B), DHFR-Petβ-helix (C), more stable DHFR-Petβ-helix D27N (D), or less stable DHFR-Petβ-helix I155A (E). Left panel, bright field images of E. coli expressing each construct. Right panel, corresponding Cy3 fluorescence images of the same field. Scale bar represents 5 μm.
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7. Figure 5
Decreasing Pertactin C-Terminal Passenger Stability, but Not N-Terminal Stability, Reduces OM Secretion
(A) Thermal unfolding of purified wild-type pertactin passenger (filled squares), N-terminal mutant W295F (open triangles), C-terminal single mutant L498K (filled triangles), and the globally-destabilizing C-terminal double mutant W423F/W440F (open circles).
(B) OM secretion of wild-type and mutant pertactin passengers. E. coli whole cell lysates were separated by SDS-PAGE and detected using an anti-pertactin polyclonal antibody. Relative percentages of processed pertactin passenger (filled arrowhead) were quantified and normalized to wild-type in each growth condition. Error bars represent the SD of three to six independent trials. Open arrowhead: unprocessed preprotein (passenger+β-domain).
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8. Figure 6
The N Terminus of the Pertactin Passenger Does Not Adopt Regular Structure
(A) Far-UV CD spectra of the entire pertactin passenger domain (triangles) and the N-terminal segment (residues 1–334) of the passenger domain (circles). The passenger domain spectrum has a minimum ∼218 nm as well as a positive signal ∼200 nm, typical for β sheet structure, whereas the N-terminal segment shows no significant contribution of α-helical or β sheet structure.
(B) Fluorescence spectra of the N-terminal segment under folding (0 M GdnHCl) and unfolding (6 M GdnHCl) conditions. The lack of significant changes for the maximum emission wavelength or total fluorescence intensity imply no significant structural changes in the protein upon addition of GdnHCl.
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9. Figure 7
Model for Outer Membrane Secretion of an Autotransporter (AT) Passenger Domain
Insertion of the C-terminal AT β-domain porin is facilitated by BamA (Jain and Goldberg, 2007; Ieva and Bernstein, 2009), but it is currently unclear whether BamA is involved in passenger transport across the outer membrane (OM). The C terminus of the passenger domain crosses the OM first (Junker et al., 2009). In vitro, the passenger C terminus is more stable than the N terminus (Junker et al., 2006) and here we have shown that reducing passenger domain C-terminal stability reduces OM secretion efficiency. In contrast, destabilizing the passenger N terminus increases OM secretion efficiency, while stabilizing the N terminus reduces secretion. These results indicate that localized regions of passenger stability, specifically a C terminus more stable than the N terminus, create an anisotropic potential that might serve as an energetic driving force for efficient OM secretion.
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